Response of the bacterial community to root exudates in soil polluted with heavy metals assessed by molecular and cultural approaches

Soil Biology & Biochemistry 32 (2000) 1405±1417

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Response of the bacterial community to root exudates in soil polluted with heavy metals assessed by molecular and cultural approaches Jacek KozdroÂj a,*, Jan Dirk van Elsas b a

b

Department of Microbiology, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland Research Institute for Plant Protection (IPO-DLO), P.O. Box 9060, 6700 GW Wageningen, The Netherlands Received 16 June 1999; received in revised form 28 October 1999; accepted 16 February 2000

Abstract We have used PCR based on 16S rDNA sequences followed by denaturing gradient gel electrophoresis (PCR-DGGE) in conjunction with cultivation-based methods to describe the e€ect of arti®cial root exudates (ARE), of which the composition simulated maize root exudates, on the structural diversity of bacterial communities in various soils di€ering in the level of contamination with heavy metals. The aim of this study was to evaluate the e€ects of organic compounds of a root exudates as a potential mechanism for selectively enhancing speci®c bacterial populations in contaminated soils, leading to the development of shifted communities di€ering in qualitative and quantitative composition. Soil microcosms were either just enriched with ARE or enriched and, additionally, ¯ooded. To characterise the response of the soil micro¯ora to the enrichment, PCR-DGGE was applied for assessment of the total bacterial community structure. Cultivation techniques were used to determine the numbers of total heterotrophic bacteria as well as of pseudomonads (which are considered to be stimulated by components of root exudates). The community structure of culturable bacteria was studied using the concept of r- and K-strategists, and isolates from dominant colonies growing on King's B agar were identi®ed by MIDI-FAME pro®ling. The results obtained showed a signi®cant e€ect of root exudates on the development of bacterial populations in soil contaminated with heavy metals. Depending on their availability and conditions prevailing in the habitat (e.g. stronger enrichment by ¯ooding) di€erent bacterial populations were stimulated, resulting in generation of di€erent community patterns by DGGE. The most signi®cant response to root exudates occurred among the culturable fraction of the soil bacteria. Distribution of bacterial classes (i.e. majority of colonies appeared after 24 h), values of EP (from 0.220 to 0.533) and CD (from 43 to 88) indices directly showed that the culturable fraction of bacteria was highly a€ected by the organic mixture simulating root exudates. These exudates reduced the bacterial diversity towards domination of r-strategists and the reduction of diversity was greater in soil with a higher contamination level. Furthermore, ¯ooding of the soils enhanced the dominance of fast growing bacteria (over 70% formed visible colonies after 24 h even on day 6) and reduced the community diversity (EP and CD indices were from about 0.291 to 0.425 and from 66 to 87, respectively). 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Microbial community structure; Root exudates; Genetic ®ngerprint; Soil enrichment

1. Introduction In heavily industrialized areas, numerous soil sites are contaminated with prohibitively high concen* Corresponding author. Fax: +48-32-2555873. E-mail address: [email protected] (J. KozdroÂj).

trations of heavy metals, which a€ect normal agricultural practices. A potential strategy to remediate these soils is the use of plants to remove pollutants from the habitat or to render them harmless (Salt et al., 1998). Recently, di€erent metal tolerant plants such as Thlaspi caerulescens, T. ochroleucum, Brassica juncea, Hordeum vulgare, Avena sativa, and others have been used for phytoextraction of Cd, Cu, Ni, Pb and Zn

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 5 8 - 4

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from contaminated soil (Ebbs and Kochian, 1997, 1998; McGrath et al., 1998; Salt et al., 1998). Successful application of plants for remediation of polluted soils depends on the phenotype and genotype of plants, but interactions between the rhizosphere micro¯ora and plant roots are also of great signi®cance. However, such microbiological aspects, to our understanding, have seldom been addressed (Burd et al., 1998; Hasnain et al., 1993; Tichy et al., 1993). In the rhizosphere, microbial biomass, activity and community structure are highly in¯uenced by speci®c physicochemical and biological characteristics prevailing in this habitat (Pearce et al., 1995; SoÈrensen, 1997). Root exudates are known as one of the most important factors a€ecting these microbiological parameters. These exudates comprise di€erent organic compounds; their amounts and composition are dependent on plant genotype, plant growth stage and environmental conditions such as CO2, light, pH, temperature, moisture and nutrients (Grayston et al., 1996). Organic acids occurring in root exudates enhance the mobilisation of several metals through weathering and chelation (Grayston et al., 1996; Vaugham et al., 1993). The bacterial communities in the rhizosphere, which can use these substrates, di€er in composition and density (SoÈrensen, 1997). However, the knowledge of microbial diversity in the rhizosphere is far from complete, since both traditional plating techniques and microscopical techniques developed to date have important limitations (Liesack et al., 1997; Madsen, 1996; Wellington et al., 1997). Therefore, the application of molecular biological techniques to detect and identify microorganisms by molecular markers, such as 16S rRNA or its corresponding gene (16S rDNA) is by far the most widely used approach to explore microbial diversity and to analyse the structure of microbial communities (Bej and Mahbubani, 1996; Head et al., 1998; Hugenholtz et al., 1998; Liesack et al., 1997; van Elsas et al., 1998). A novel method, PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE), was recently proposed for studying complex microbial populations (Muyzer et al., 1993). In this method, total microbial DNA is extracted from soil and then the bacterial 16S rDNA is ampli®ed by PCR with universal eubacterial primers. The PCR-ampli®ed 16S rDNA fragments of the same length but with di€erent sequences can be separated in polyacrylamide gels containing a linearly increasing gradient of denaturants. The patterns obtained by DGGE or the related technique, temperature gradient gel electrophoresis (TGGE), provide information about the structural diversity of bacterial groups, including the nonculturable ones (Muyzer and Smalla, 1998). Recently, Griths et al. (1999) using community DNA hybridisation, % G + C pro®ling and phospho-

lipid fatty acid analysis (PLFA), showed signi®cant changes in microbial community structure in response to synthetic root exudates, which were applied continuously to a soil held at constant water potential. The microbial community structure changed consistently as substrate loading increased, and fungi dominated over bacteria at high substrate loading rates. Bossio and Scow (1995) showed that carbon inputs into soil and ¯ooding increased counts of active bacteria and respiration rates, but decreased the metabolic diversity of bacterial populations. Despite these observations, the knowledge about relationships between bacterial community and root exudates is still limited. In addition, the possible roles of rhizosphere processes in phytoremediation of polluted soil are poorly understood. In this study, we used PCR-DGGE in conjunction with cultivation-based methods to describe the e€ect of arti®cial root exudates (ARE), which mimicked maize root exudates, on the structural diversity of bacterial communities in various soils di€ering in the level of contamination with heavy metals. The aim of this study was to evaluate the action of the organic compounds in root exudates as a potential mechanism for selective enhancement of bacterial populations in contaminated soils, leading to the development of novel communities. These root-dependent changes in the soil micro¯ora composition may be an essential process in successful phytoremediation of soils polluted with heavy metals. The remediation activity that is thought to be associated with plants is often due to the activity of plant-associated microorganisms (O'Connell et al., 1996). 2. Materials and methods 2.1. Soil Composite soil samples (each prepared from eight di€erent randomly-collected cores) were collected from the surface (0±10 cm) at two sites (PS1 and PS19) of Piekary Slaskie in Silesia, a highly industrialized region of Poland. The soil with low contents of heavy metals was the silt loam PS1 (37% sand, 54% silt, 11% clay, 3.4% organic C, pHKCl 6.2, 160 mg Pb kgÿ1, 4.4 mg Cd kgÿ1, 330 mg Zn kgÿ1). The more contaminated soil was silt loam PS19 (37% sand, 54% silt, 10% clay, 4.6% organic C, pHKCl 6.3, 1830 mg Pb kgÿ1, 23.3 mg Cd kgÿ1, 2390 mg Zn kgÿ1). Concentrations of heavy metals were determined by atomic absorption spectrophotometry after soil extraction with aqua regia (conc. HNO3/conc. HCl, 1:3, vol/vol). All soil samples were sieved (2 mm-mesh) and stored moist at 48C prior to use. For microcosm experiments, two air-dried (about 10% moisture content) 100 g portions of PS1 and PS19 were placed into 300 ml Erlenmeyer ¯asks

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and wetted with either 20 ml of ARE or distilled water (control), establishing a moisture content equal to 70% WHC (Alef and Nannipieri, 1995). One set of soils was additionally ¯ooded with 35 ml of distilled water, receiving a 50-mm layer of water over the soil. The ¯asks were covered with para®lm and incubated at room temperature, under gentle shaking (80 rpm) in case of the ¯ooded microcosms. The ARE were based on the sugar, organic acid and amino acid composition of root exudates of maize (Kra€tczyk et al., 1984). The concentrations of particular components of ARE were recalculated from the data of Kra€tchyk et al. (1984), considering their contents per 1 g of soil contained in 1 mm zone of the rhizosphere (Table 1). 2.2. DNA analysis DNA was extracted from 1-g soil samples after 1, 3 and 6 days of PS1 and PS19 incubation by the method of Saano and Lindstrom (1995). Puri®cation of the crude extracts was by CsCl and potassium acetate precipitation steps followed by Wizard (Promega, USA) spin column treatment (van Elsas et al., 1997). Absorbency measurements at A260 and A280 were determined with a GeneQuant RNA/DNA calculator (Pharmacia, Sweden) and a small-volume quartz cuvette to calculate the concentration (1 A260 unit ˆ 50 mg mlÿ1 double-stranded DNA) and the A260/A280 purity ratio of DNA samples (Crecchio and Stotzky, 1998). DNA quality (size) was checked by electrophoresis in 0.8% (wt/vol) horizontal agarose gel run in 0.5% TBE bu€er and stained with 0.9 mg mlÿ1 of ethidium bromide (Sambrook et al., 1989). A 1-ml volume (roughly 5±10 ng) of each extracted DNA was ampli®ed by PCR with a Peltier thermal cycler PTC 200 (MJ Research, USA). The PCR mixture used contained 0.2 mM of each primer, 200 mM of each dNTP, 5 ml of 10  Sto€el bu€er (Perkin-Elmer, USA), 5 U of AmpliTaq Sto€el fragment (PerkinTable 1 Composition of arti®cial root exudates (ARE) of maize based on Kra€czyk et al. (1984) Sugars (mg gÿ1)a

Organic acids (mg gÿ1)

Amino acids (mg gÿ1)

Glucose Arabinose Fructose Saccharose

Oxalic acid Fumaric acid Malic acid Citric acid Succinic acid Benzoic acid Tartaric acid Glutaric acid

Glutamic acid Proline Alanine Glycine Leucine Serine Arginine Glutamine Valine

7.080 2.760 2.340 1.530

1.960 3.660 0.260 0.460 0.310 0.190 0.070 0.030

0.068 0.061 0.057 0.037 0.017 0.025 0.030 0.012 0.010

a All component concentrations were calculated per 1 g of soil contained in 1 mm zone of the rhizosphere.

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Elmer, USA), 3.75 mM MgCl2, 0.5 ml of 1% (vol/vol) formamide, 0.25 mg T4 gene 32 protein (Boehringer, Mannheim, Germany) and sterile Milli-Q water to a ®nal volume of 50 ml. The primers for PCR were speci®c for conserved bacterial 16S rDNA sequences (Heuer and Smalla 1997). PCR with primers R1401 (5 ' GCG TGT GTA CAA GAC CC-3 ') and F968GC (5 ' GC clamp-AAC GCG AAG AAC CTT AC-3 ') ampli®ed a bacterial 16S rDNA fragment from positions 968 to 1401 (Escherichia coli numbering). The GC-rich sequence attached to the 5' end of primer F968GC prevents complete melting of the PCR products during subsequent separation on the denaturing gradient during DGGE (Muyzer et al., 1993). PCR ampli®cation was performed for 40 thermal cycles in a touchdown scheme (Duarte et al., 1998) as follows: after initial denaturation of 4 min at 948C, each cycle consisted of denaturation at 948C for 1 min, primer annealing at TA for 1 min, and primer extension at 728C for 1 min. In the ®rst 10 cycles, TA decreased by 28C every second cycle from 658C in the ®rst cycle to 578C in the 10th. In the last 30 cycles, TA was 558C. Cycling was followed by ®nal primer extension at 728C for 10 min. PCR products were visualised by electrophoresis in 1.2% (wt/vol) agarose gels after ethidium bromide (0.9 mg mlÿ1) staining (Sambrook et al., 1989). Strong bands of the expected size (450 bp) were subjected to DGGE analysis. DGGE (Muyzer et al., 1993; Heuer and Smalla, 1997) was performed with an Ingeny phorU-2 system (Leiden, Netherlands). Samples of 20 ml of PCR product were loaded onto 6% (wt/vol) polyacrylamide gels in 0.5  TAE bu€er. The polyacrylamide gels were made with a denaturing gradient ranging from 45% at the top of the gel to 65% at the bottom (where 80% denaturant contains 5.6 M urea and 32% formamide). The electrophoresis was run for 16 h at 608C and 100 V. After the runs, gels were removed from the setup and stained for 60 min with SYBR green I nucleic acid gel stain (Molecular Probes, Netherlands). The stained gels were immediately photographed on an UV transillumination table with a CCD camera and scanned (Biozym, Netherlands). Digital images of the gels showed banding patterns that were analysed by the 1/0 clustering method of the NT-SYS program (Exeter Software, USA) by using the unweighted pair group with mathematical averages (UPGMA). This method allowed the construction of dendrograms that show clustering trends among the soil samples analysed. 2.3. Culturable fraction analysis Soil samples (5 g) were collected after 1, 3 and 6 days of incubation and placed in Erlenmeyer ¯asks containing 45 ml of 0.1% sterile NaPP (pH 7.0) and 5 g of gravel. The ¯asks were shaken at 200 rpm for 30

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min. Serial 10-fold dilutions of the soils in 0.85% NaCl were plated in duplicate on 0.1-strength TSA for total bacterial counts (De Leij et al., 1993; van Elsas et al., 1994), and numbers of pseudomonads were counted on King's B agar (Difco proteose peptone no. 3 20 g, K2HPO4
3H2O 1.8 g, MgSO4
7H2O 1.5 g, 87% glycerol 15 ml, agar 15 g, demineralized water 1 `, pH 7.2). Plates were incubated at 278C for 10 days, and cfu numbers of the bacteria studied were determined after 6 days. The bacterial community structure was characterised following the method described by De Leij et al. (1993). Colonies on 0.1-strength TSA were enumerated on a daily basis for ®ve times and, in addition, on day 10. This way, several classes of culturable organism were generated per plate. The number of bacteria in each class was expressed as a proportion (%) of the total count. The di€erent distributions of the classes gave an insight into the distribution of r- and K-strategists in each sample. R-strategists are de®ned here as fast growing bacteria forming visible colonies within 24 h in response to enrichment, while K-strategists are characterised by slow growth and colonies are produced later. To characterise the community composition in the form of a single value two indices were calculated. The eco-physiological (EP) index proposed by De Leij et al. (1993) was calculated according to the following equation: H 0 ˆ ÿS…pi  log pi †, where pi represents each of the six classes de®ned above as a proportion of the total population in the soil sample ( pi-population in class i per total population). The more even the distribution of the classes, the higher the EP-index. The colony development (CD) index proposed by Sarathchandra et al. (1997) was calculated as follows:

washing (Operating manual version 6.0, MIDI, USA). The organic phase containing cellular FAME was separated by Hewlett Packard 6890 GC on a capillary column Ultra 2-HP (cross-linked 5% phenyl±methyl silicone; 25 m, 0.22 mm ID; ®lm thickness, 0.33 mm) with hydrogen as the carrier gas and analysed by Sherlock MIS software, using the aerobe method and TSBA library version 3.9 (MIDI, USA). 2.4. Statistics Prior to the DNA and cultural fraction analyses each duplicate soil microcosm was sampled, and data of bacterial counts (log cfu), EP and CD indices were expressed as the means and treated statistically by oneway ANOVA (Statistica 7.0 PL). Since both the duplicates represented virtually identical pictures after PCRDGGE, the one showing better pattern was chosen for presentation and a dendrogram drawing. 3. Results Former work with di€erent methods of DNA extraction from soil contaminated with heavy metals showed that the direct method of bacterial cell lysis with proteinase K at 378C (Saano and Lindstrom, 1995) and further extraction and puri®cation of the crude extract yielded DNA pure enough for molecular analysis with relatively high eciency. Therefore, this method was chosen for DNA extraction from the PS soils selected in this study. Two Silesian soils, i.e. PS1 and PS19, similar in structure and chemical characteristics, but di€erent in concentrations of heavy metals, were used for the experiments. The addition of ARE to the PS soils or additional enhancement of microbial growth in the enriched soils by ¯ooding did not increase the yields of extracted DNA (Table 2). After puri®cation, DNA was colourless and showed A260/

CD ˆ …N1 =1 ‡ N2 =2 ‡ N3 =3 ‡ . . . ‡ N10 =10†  100, where N1, N2, N3, . . .,N10 represent the proportion (i.e. bacterial colonies appearing on each counting day expressed as a proportion of the total number of colonies appearing over the 10 day period) of bacterial colonies appearing on days 1, 2, 3, . . ., 10. The more even the distribution of the classes is, the lower the CD-index will be. Dominant colonies growing on King's B agar were selected and identi®ed based on whole-cell cellular fatty acids, derivatized to methyl esters, i.e. FAME, and analysed by gas chromatography (GC), using the MIDI system (Microbial Identi®cation System, USA). FAME were extracted from each isolate using the standard and recommended procedure, consisting of saponi®cation, derivatization, extraction and ®nal base

Table 2 Comparing yield of DNA (mg gÿ1 soil)a extracted from polluted soils amended with arti®cial root exudates (ARE)b Soil + treatment

Day 1

Day 3

Day 6

PS1 + ARE PS1 + ARE + ¯ooding PS1 PS19 + ARE PS19 + ARE + ¯ooding PS19

72.8 86.8 53.1 39.4 34.7 26.6

59.5 (36.7) 51.1 (28.3) NDc 22.4 (11.7) 25.9 (3.1) NDc

36.4 (11.4) 13.1 (9.2) NDc 20.1 (19.9) 36.2 (3.0) NDc

a

(28.4) (29.3) (15.4) (23.8) (0.4) (1.3)

Assessed via A260 measurement. Values are means and standard deviations in the parenthesis. c ND Ð DNA contents were not determined because we assumed that DNA yields did not change in the nonenriched soils during several days. b

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A280 values of 1.6±1.7. These DNA samples were suciently pure for subsequent PCR-DGGE analysis. PCR ampli®cation of 16S rDNA fragments successfully generated 450 bp products visible as strong bands in the gel after electrophoresis (data not shown).

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Samples collected from the soils enriched with ARE or enriched and additionally ¯ooded showed variations in banding patterns when analysed by PCR-DGGE (Fig. 1). In all patterns, 24±31 bands of various intensities were detected per sample, with about 15 bands

Fig. 1. DGGE patterns of 16S rDNA fragments ampli®ed with DNA from soils polluted with heavy metals. The soils were either enriched with ARE or enriched and additionally ¯ooded. (A) Untreated soils; lane 1, community pattern of PS1, lane 2, community pattern of PS19. (B) Enriched soils; lanes 1±4, community patterns on day 6 of PS19 + ARE + ¯ooding, PS1 + ARE + ¯ooding, PS19 + ARE and PS1 + ARE, respectively; lanes 5±8, community patterns on day 3 of PS19 + ARE + ¯ooding, PS1 + ARE + ¯ooding, PS19 + ARE and PS1 + ARE, respectively; lanes 9±12, community patterns on day 1 of PS19 + ARE + ¯ooding, PS1 + ARE + ¯ooding, PS19 + ARE and PS1 + ARE, respectively. Percent values indicate the percentage of denaturant at each position.

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shared among all samples. Generally, 37 band positions were recognised in the DGGE gel. The highest number of bands (31) was detected in the ¯ooded PS19 on day 3. The other pro®les were not signi®cantly di€erent as to the number of bands detected. In the control soils, 23 and 27 bands were found in PS1 and PS19, respectively. The di€erence in band intensity was presumed to indicate numerical di€erences between the target molecules. Clustering of the pro®les revealed that all pro®les were about 78% similar, with some trends with respect to the clustering above this level (Fig. 2). One day after soil enrichment with ARE, the pro®les of PS1 and PS19 were almost identical (99% similarity). However, when ¯ooded, PS1 and PS19 pro®les belonged to di€erent clusters. On day 6, they showed 91% similarity and formed one cluster (Fig. 2). The pro®les were the most di€erent on day 3 when they did not show any trend with respect to the clustering. At the end of soil microcosms' incubation, PS1 and PS19 pro®les formed two distinct clusters comprised of either the ¯ooded soils or those only enriched with ARE. Clustering of the banding patterns was not associated with concentrations of heavy metals in the soils studied (Fig. 2). The enrichment of the contaminated soils with ARE or additional ¯ooding generated banding pro®les that maintained 71% similarity to the pro®les representing the bacterial communities of the untreated soils. The dendrogram represents only the qualitative similarity between the banding pro®les. This similarity does not consider the intensities of bands. Each pro®le showed several di€erent bands of high intensity. On

day 6, ¯ooding of the enriched PS1 and PS19 soils led to the appearance of ®ve and two dominant bands in the pro®les (at around 53±56% denaturant), respectively. In addition, two distinct bands (at around 50± 51% denaturant) were found in the ¯ooded PS19 after 6 days of incubation (Fig. 1). A single intensive band around 62% denaturant was detected in the pro®le of ARE enriched PS19 on day 3. This band, albeit less intensive, was also present in the pro®les of enriched PS1 and PS19 after 6 days of incubation (Fig. 1). Determination of counts of culturable bacteria showed a signi®cant positive e€ect of soil enrichment with ARE under unsaturated and ¯ooded conditions. The numbers of total heterotrophic bacteria did not di€er signi®cantly between PS1 and PS19 when the soils were enriched with ARE or left untreated (Fig. 3). On day 6, the cfu counts decreased in ARE treated PS1 and PS19. When these soils were ¯ooded, cfu counts increased signi®cantly compared to the un¯ooded conditions. In the PS1 soil, cfu counts of total heterotrophs increased during incubation with ARE after ¯ooding (Fig. 3). In contrast, the cfu numbers decreased in PS19 on day 6. Hence, the numbers of total heterotrophic bacteria were highest in ¯ooded PS19 on day 1 but in ¯ooded soil PS1 on day 6. Similar trends in cfu counts were found for the bacteria growing on King's B agar (Fig. 4). However, signi®cant di€erences between cfu counts in un¯ooded PS1 and PS19 soils enriched with ARE were found on day 6. This was in contrast with the results of the bacterial counts determined on 0.1-strength TSA. Representatives of bacteria that formed the highest number of colonies on King's B agar were isolated

Fig. 2. Genetic similarity of microbial-community pro®les obtained with PCR-DGGE from soils contaminated with heavy metals. The soils PS1 and PS19 were either enriched with ARE or enriched and additionally ¯ooded. 1±4, the communities on day 6 in PS19 + ARE + ¯ooding, PS1 + ARE + ¯ooding, PS19 + ARE and PS1 + ARE, respectively; 5±8, the communities on day 3 in PS19 + ARE + ¯ooding, PS1 + ARE + ¯ooding, PS19 + ARE and PS1 + ARE, respectively; 9±12, the communities on day 1 in PS19 + ARE + ¯ooding, PS1 + ARE + ¯ooding, PS19 + ARE and PS1 + ARE, respectively; 13, the community in PS1 untreated; 14, the community in PS19 untreated.

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and identi®ed by MIDI-FAME pro®ling. Dominant isolates from both PS soils were identi®ed as Comamonas acidovorans. In PS1 enriched with ARE, Arthrobacter oxydans and Stenotrophomonas maltophilia dominated, whereas Variovorax paradoxus and C. acidovorans were isolated from ARE enriched PS19. When enriched PS1 and PS19 were additionally ¯ooded, the dominant isolates were identi®ed as V. paradoxus, Pseudomonas putida and C. acidovorans, respectively. Colonies of bacteria that developed on 0.1strength TSA were generally represented by fastgrowing organisms when they were isolated from both ARE enriched or additionally ¯ooded PS soils (Fig. 5). By contrast, higher numbers of visible colonies were formed from 48 h onwards, when isolated from the unamended control soil. In the lightly contaminated PS1 soil, a shift towards slowgrowing bacteria was found over time when the soil was treated with ARE (Fig. 5). When the soil was additionally ¯ooded, populations that formed visible colonies after 24 h were dominating during incubation of the soil. In contrast, bacteria forming colonies after 24 h dominated in PS19 enriched with ARE after 3 and 6 days of the soil incubation, while they constituted only 35% on day 1 (Fig. 5). Compared to the untreated PS1, higher

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numbers of colonies were revealed after 48 h when PS19 was analysed. Calculation of EP indices showed that the enrichment of soil with ARE or additional ¯ooding decreased the EP values as compared with those of the unamended controls (Table 3). For the soils enriched with ARE under un¯ooded conditions, EP values were signi®cantly lower for total heterotrophic bacteria in PS19 than in PS1. With the exception of the data obtained on day 3, similar trends were observed for the soils that were ¯ooded. However, the di€erences between PS1 and PS19 were either small or not signi®cant. Generally, EP indices of the bacterial populations from ¯ooded soils were lower than those from the soils only enriched with ARE (Table 3). CD values of the bacterial community on 0.1strength TSA were signi®cantly higher for both the ARE enriched and the treated and subsequently ¯ooded soils compared with the untreated soils. Total heterotrophic bacteria in treated and untreated soil PS19 showed higher CD values than those of PS1, especially on day 3 and 6 (Table 3). Compared to the EP indices, signi®cant di€erences between CD values of the bacterial community originating from ¯ooded PS1 enriched with ARE were found during incubation. The same observation was found for bacteria isolated from ARE enriched PS19, when CD values obtained

Fig. 3. Numbers (log cfu gÿ1 dry soil) of culturable heterotrophic bacteria in soils polluted with heavy metals. The numbers were determined after 6 days of incubation at 278C. The soils PS1 and PS19 were either enriched with ARE or enriched and additionally ¯ooded. (A) Soil + ARE; (B) soil + ARE + ¯ooding; (C) control.

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Fig. 4. Numbers (log cfu gÿ1 dry soil) of culturable bacteria growing on King's B agar isolated from soils polluted with heavy metals. The numbers were determined after 6 days of incubation at 278C. The soils PS1 and PS19 were either enriched with ARE or enriched and additionally ¯ooded. (A) Soil + ARE; (B) soil + ARE + ¯ooding; (C) control.

on day 1 and 6 were compared (Table 3). Generally, CD values of the bacterial community were higher for ¯ooded soils compared to the soils that were only enriched with ARE. 4. Discussion The excretion of di€erent organic compounds and the sloughing o€ of root hairs and epidermal cells are major factors responsible for the stimulation of micro-

organisms in the rhizosphere (Pearce et al., 1995; SoÈrensen, 1997). The excreted compounds are components of root exudates, which play an important role in carbon ¯uxes into soil, a process also called rhizodeposition (Grayston et al., 1996; Pearce et al., 1995). The rhizodeposition e€ect was studied in this work, in which a mixture of sugars, organic acids and amino acids was added to polluted soils. To enhance the possible e€ect of these ARE on the indigenous soil micro¯ora, and to make soil conditions more similar to those of a rhizosphere in water-logged conditions

Table 3 Values of eco-physiological (EP) and colony development (CD) indices of culturable heterotrophic bacteria isolated from soils contaminated with heavy metals and enriched with arti®cial root exudates (ARE)a Soil

Treatment

Day 1 EP

PS1 PS19

a

ARE ARE + ¯ood Control ARE ARE + ¯ood Control

0.492 0.425 0.616 0.362 0.401 0.486

Day 3 CD

(0.029) (0.033) (0.016) (0.001) (0.008) (0.013)

69.2 65.7 32.3 65.9 78.0 43.7

EP (2.2) (0.4) (1.8) (2.3) (0.2) (3.1)

Values are the means and standard deviations in the parenthesis.

0.533 0.436 0.618 0.220 0.467 0.482

Day 6 CD

(0.016) (0.021) (0.024) (0.028) (0.039) (0.011)

55.0 78.1 33.4 87.8 70.7 42.9

EP (2.2) (1.8) (1.5) (2.6) (0.4) (3.0)

0.636 0.394 0.620 0.377 0.291 0.484

CD (0.025) (0.051) (0.020) (0.020) (0.006) (0.015)

43.4 82.4 32.8 78.2 86.9 43.9

(0.9) (4.3) (2.0) (0.4) (1.9) (2.6)

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(e.g. reduced oxygen potential, permanent easy access to nutrients), the soils were ¯ooded and incubated as slurries. The total yields of DNA extracted from these soils did not change in response to soil enrichment, however, the numbers of culturable heterotrophic bacteria increased signi®cantly. This is contradictory to the statement that concentrations of microbial DNA in rhizosphere and bulk soil is related to microbial cell densities, especially when the DNA content of microbial cells can be described using an average number (Leung et al., 1995). However, for estimates of DNA yields obtained from the rhizosphere it is important to note that di€erent bacterial species have di€erent genome sizes as well as di€erent lysis characteristics (Trevors, 1996). Hence, sometimes obscure and variable relationships between cell numbers and DNA yields have been found (Leung et al., 1995). The concentrations of heavy metals in polluted soils also did not have an e€ect on bacterial counts and, consequently, DNA yields and purity. This could result from the fact that both soils (i.e. PS1 and PS19), containing di€erent concentrations of total heavy metals, likely characterised similar contents of bioavailable forms. In contrast, Griths et al. (1997) reported that soils contaminated with heavy metals, especially Pb, Zn and Cd, charac-

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terised by a lower microbial biomass and DNA yields, compared to the uncontaminated control. However, DNA extracted per unit of microbial biomass C was increased in Cu and Ni contaminated soil, and decreased by Pb, Zn and Cd. The authors did not ®nd any correlation between DNA yields and microbial biomass. Extractability of DNA may depend on the type and physiological status of the microorganisms present, and these factors may depend on the history of soil contamination. Hence, a signi®cant e€ect of the contamination on soil micro¯ora should be expected even if no di€erences in culturable bacterial counts are found. The results obtained with PCR-DGGE showed that soil enrichment with ARE slightly shifted the abundance of major bacterial groups (populations), especially when the soil was additionally ¯ooded. Root exudates are known to a€ect microbial density and species richness (SoÈrensen, 1997; Tate, 1995). However, when analysing the whole bacterial community at molecular level, the observed e€ect of exudates may be small or undetectable. Duineveld et al. (1998) using PCR-DGGE found rather similar bacterial communities between the chrysanthemum rhizosphere and bulk soil. Moreover, the DNA-based ®ngerprints indicated that the bacterial community structure, compris-

Fig. 5. Community structure of culturable fraction of bacteria in soil contaminated with heavy metals. The soils PS1 and PS19 were either enriched with ARE or enriched and additionally ¯ooded. (A) Soil + ARE; (B) soil + ARE + ¯ooding; (C) control. Fast growing bacteria (rstrategists) form visible colonies within 24 h, and slow growing bacteria (K-strategists) form colonies later.

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ing several dominant groups, was stable in soil and the rhizosphere both in time and space. The authors suggested that the potential impact of the root on bacterial populations in soil was not strong enough to induce major shifts in community structure. The dominance of a reduced number of bacterial populations was also found in the rhizosphere of potato after analysing of TGGE pro®les (Heuer and Smalla, 1997). In contrast, the presence of speci®c organic compounds in root exudates may even decrease the microbial diversity in the rhizosphere leading to the dominance of a few species. This was shown by Smalla et al. (1998) when 16S rDNA TGGE pro®les of potato rhizosphere bacterial communities that had responded to di€erent organic substrates of BIOLOG reaction wells were compared with those of the untreated control. In this study, soil enrichment with ARE not only a€ected the number of dominant bacterial types but also caused changes in the structural diversity. The soils contaminated with di€erent concentrations of heavy metals (i.e. PS1 versus PS19) could be characterised by bacterial communities that showed a more similar composition when the soils were treated in the same way than when the same soil was treated with ARE under di€erent conditions. This indicates that the structural diversities of bacterial communities were relatively similar in these soils. However, the reaction of soil bacteria to the mixture of organic compounds, simulating the rhizodeposition e€ect of root exudates, was dependent on the ``strength'' of the enrichment. When the soils were ¯ooded, organic compounds of ARE were probably more available for the soil bacteria. In contrast, ARE might have been sorbed on soil particles after addition to un¯ooded soil, resulting in a di€erent response of the indigenous bacteria. Under un¯ooded conditions, a fast reaction of soil bacteria to the enrichment with ARE could be observed, whereas ¯ooding resulted in the predominance of fast growing bacteria over longer time, especially in the lower polluted soil. This way, more bacterial populations responding to root derived compounds could be detected. Duineveld et al. (1998) reported that the e€ect of roots on dominating soil bacterial groups was marginal because these bacteria were probably oligotrophic and responded slowly to the changes brought about by root exudates. They stated that each soil type may have its typical set of dominant bacterial groups, and this mainly determines the DNA-based pro®les of bacterial communities obtained in the rhizosphere. In contrast, Maloney et al. (1997) applying a physiological approach, found that the qualitative and quantitative composition of root exudates strongly a€ected the community structure of lettuce and tomato rhizospheres. Their results agree with the DNA-based ®ngerprints of the present study. However, the development of the microbial

community in the rhizosphere depends on soil type, plant species, plant growth stage, presence of contaminants, temperature and other environmental factors (Anderson et al., 1995; Pearce et al., 1995). The composition of root exudates a€ects the structural diversity of bacterial communities not only qualitatively but also quantitatively. Di€erent species (populations) of bacteria were positively a€ected by ARE under un¯ooded and ¯ooded conditions. This indicates that a longer access to the organic compounds of ARE could stimulate the development of speci®c bacterial populations living in micropores, where these substrates become available after ¯ooding. However, the stronger intensity of some bands in the DGGE pro®les could be associated not only with higher numbers of speci®c bacterial species. Since one band may represent more than one species, the increase of some bands' intensity could be connected with the detection of higher number of di€erent species with similar rDNA sequences, which were stimulated by ARE (Heuer and Smalla, 1997). These populations represented by dominant bands may be of special interest, when bacterial species that speci®cally respond to root exudates in a heavy metal containing background are to be obtained. A common reaction of the rhizosphere micro¯ora to organic compounds exuded by plant roots is an increase in the numbers of culturable cells (Grayston et al., 1996). This result was also found in this study. Two soils contaminated with di€erent concentrations of heavy metals showed similar numbers of total heterotrophic bacteria. However, di€erences were revealed when ARE enriched soils were additionally ¯ooded, which increased substrate availability for indigenous bacteria. In comparison with PS1 on day 6, the lower numbers of bacteria in PS19 could result from the impact of higher concentrations of heavy metals released from soil colloids during incubation. Organic acids contained in ARE and those produced by intensively multiplying bacteria in response to added ARE, may have leached heavy metals into soil solution, increasing their availability (Banks et al., 1994; Ehrlich, 1997; Jones and Darrah, 1994; Krishamurti et al., 1996). Gram-negative copiotrophic organisms such as pseudomonads may be strongly stimulated in the rhizosphere by root exudates. Results of this study clearly showed that this group dominated among culturable heterotrophic bacteria in both enriched soils. The addition of ARE to the PS soils induced the development of other pseudomonads than Comamonas acidovorans that dominated in untreated soils. In addition, a Gram-positive isolate, Arthrobacter oxydans, appeared in PS1 amended with ARE. The isolates identi®ed in this study were also detected by other authors in various contaminated soils. Arthrobacter-like strains

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dominated among culturable bacteria isolated from soil contaminated with Zn (Heuer and Smalla, 1997). Heavy metal resistant representatives of Arthrobacter and Alcaligenes (Variovorax ) were found in highly lead-contaminated sites (Trojanovska et al., 1997). Di Giovanni et al. (1996) isolated 2,4-dichlorophenoxyacetic acid-degrading Variovorax paradoxus from a contaminated soil. De Leij et al. (1993) proposed a simple method based on the growth response when exposed to substrate for determination of the community structure of the culturable fraction of soil micro¯ora. The method applied in this study showed that both polluted soils enriched with ARE under un¯ooded and ¯ooded conditions were dominated by fast growing rstrategists that are characteristic for environments rich in easily available nutrients. In contrast, the soils unamended with ARE were dominated by K-strategists characteristic for nutrient-poor and uncrowded habitats. Fast growing bacteria (r-strategists) are often predominant on young immature roots and in the rhizosphere, where a high release of readily available growth and energy substrates occurs (Chiarini et al., 1998; De Leij et al., 1993; Nacamulli et al., 1997; Sarathchandra et al., 1997). The possible faster disappearance of ARE in PS1 due to microbial activity could explain the shift in community structure towards K-strategists observed on day 6, compared with microbial community in PS19. The level of soil contamination with heavy metals could be a major factor in this process. The e€ect of the metal concentrations on culturable bacterial populations in soil was evident when EP indices were calculated. A lower diversity was found in PS19 than in PS1, especially when the soil was not ¯ooded. The uneven distribution of classes within the culturable bacterial community in contaminated soils amended with ARE was also con®rmed by CD indices. In addition, this index indicated further reduction of diversity among r-strategists, while the EP indices showed similar values, suggesting no changes were occurring in bacterial community. Furthermore, Sarathchandra et al. (1997) found that the CD-index was of greater relevance, and related better to the r±K concept than the EP-index. Generally, both indices directly showed that the culturable fraction of bacteria was highly a€ected by the organic mixture simulating root exudates. These exudates reduced bacterial diversity towards domination of r-strategists and the reduction of diversity was greater in the higher contaminated soil. Also, ¯ooding of the soils enhanced the dominance of fast growing bacteria and reduced the community diversity. The results obtained in this study clearly showed signi®cant e€ect of root exudates on the development of bacterial populations in soil contaminated with heavy

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metals. Depending on their availability and conditions prevailing in the habitat (e.g. stronger enrichment by ¯ooding) various bacterial populations can be stimulated resulting in the generation of di€erent community patterns. The stimulation of speci®c populations in contaminated soil by organic substrates exuded by plant roots may be an important result of phytoremediation. Identifying of these species by reampli®cation, sequencing and/or hybridisation with speci®c probes would be an interesting endeavour in this strategy. These isolates, if metal-resistant or metal-transforming could be studied for a plant growth promoting activity and might be useful for reintroduction into polluted soil and use in remediation processes. The other approach based on the application of a consortium of microorganisms actively responding to root exudates and indicating a remediative capacity is also worth considering. It is dicult to predict whether such approaches result in the reduction of microbial diversity in the rhizosphere and soil. Nevertheless, selected species or a consortium of species can stimulate phytoremediation directly by immobilization of heavy metals, reducing amount of bioavailable metals that could be toxic for plants. In addition, an indirect stimulation of phytoremediation by the bacteria used likely occur due to the bacterial-mediated stimulation of plant growth. Thus, the plant can accumulate higher amounts of heavy metals inside tissues and/or enhance immobilization of the metals outside due to increased exudation of di€erent organic compounds by roots. The results of this study indicate that the most signi®cant response to root exudates occurred among the culturable fraction of soil bacteria. However, this does not imply that these bacteria will be the dominant ones in the rhizosphere (Duineveld et al., 1998). Comparison between total community pro®les and those of the culturable fractions obtained by PCR-DGGE showed only a few bands in common (Heuer and Smalla, 1997). Culturable bacteria are considered to represent only a small fraction of the rhizosphere micro¯ora, which, however, can very quickly respond to root exudates (Duineveld et al., 1998). This study indicates that root-derived organic compounds will signi®cantly a€ect the development of the rhizosphere micro¯ora. This impact may be very important in order to restore biological components of contaminated soil to the form characteristic for an agriculturally productive soil. Further studies that are more precise are needed to assess the potential role for bacteria in phytoremediation of the heavy metal polluted soils. The interactions between the root exudates and soil micro¯ora occurring in the rhizosphere seem to be key for successful application of plants in bioremediation of polluted sites.

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Acknowledgements This work, except identi®cation of bacterial isolates, was performed with ®nancial support for Jacek KozdroÂj by a ``MOE'' grant to work in the laboratory of Jan Dirk van Elsas at IPO-DLO, Wageningen. The authors thank Ludwina Lankwarden and Anneke Keijzer-Wolters for valuable technical assistance.

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